1. Field of the Invention
This invention relates to novel valproic acid derivatives pertaining to binding assays, especially immunoassays, for determining valproic acid and its salt forms in liquid media such as serum. Such derivatives include labeled valproic acid conjugates directly used in performing such assays. Also provided are antibodies to valproic acid and immunogen conjugates useful in stimulating production of such antibodies in host animals according to conventional techniques. Further provided are intermediates in the synthesis of the aforementioned labeled conjugates and immunogen conjugates.
Valproic acid (2-n-propylpentanoic acid) and its various salt forms, particularly its sodium salt, are anticonvulsant drugs useful in the management of epilepsy. The structural formula for the acid is as follows: ##STR1## cf. The Merck Index, 9th ed., no. 9574(1976).
Due to its rapid elimination, blood levels of valproic acid fluctuate considerably. There is only a poor correlation between daily dose and blood concentration, possibly due to interindividual differences in absorption or total body clearance. Therefore, the proper management of epilepsy with the drug cannot be achieved by the simple choice of a medication regimen based on body weight, surface area, or age of the patient. Frequent administration and determination of individual blood concentrations at accurately fixed times appear to be the only reliable monitoring method. No clear therapeutic blood levels have been firmly established. However, some physicians accept 50 to 100 .mu.g/ml of valproic acid as the range of therapeutic blood level since most patients with blood levels in this range seem adequately controlled [Pinder et al, Drug 13:94(1977)].
2. Description of the Prior Art
At present valproic acid is usually analyzed by gas-liquid chromatography [Libeer et al, J. Chromatogr. 160:285(1978)], with high-pressure liquid chromatography also being used [Sutheimer et al, Chromatogr. Newsletter 7:1(1979)]. There is presently no commercially available immunoassay for valproic acid. An abstract has been published regarding a homogeneous enzyme immunoassay for valproic acid [Clin. Chem. 25:1093(1979)]. No details were given concerning any of the reagents which might be used in such an assay.
Nonradioisotopic specific binding assays employing an enzyme-cleavable substrate label are described in German Offenlegungschriften Nos. 2,618,419 and 2,618,511, corresponding respectively to U.S. Patent Applications Ser. Nos. 667,982 and 667,996, both filed Mar. 18, 1976, and assigned to the present assignee. The assays avoid the use of radioisotopic labels and can be performed in homogeneous or heterogeneous formats. In the heterogeneous format, the bound- and free-species of the labeled conjugate are physically separated and the label measured in one of the separated species, whereas in the homogeneous format, the label expresses a different activity in the bound-species compared to the free-species, permitting performance of the assay without a separation step. In the aforementioned assays, the labeled conjugate serves as a substrate for a cleaving enzyme, with cleavage of the conjugate producing a distinguishable indicator product, usually a fluorescent product. The fluorescers umbelliferone or fluorescein are coupled to the ligand under assay through an ester bond which upon cleavage by an esterase releases the free fluorescent products, umbelliferone and fluorescein, respectively.
An improved substrate-labeled specific binding assay is described in pending U.S. Patent Application Ser. No. 886,094, filed Mar. 13, 1978, and the continuation-in-part application Ser. No. 87819 based thereon filed on or about Oct. 19, 1979, both assigned to the present assignee, and by Burd et al, Clin. Chem. 23:1402(1977). The improvement comprises employing as the label component of the labeled conjugate, a residue of the formula "G-D-R" wherein G is a glycone, D is a dye indicator moiety, and R is a linking group through which the dye indicator moiety is covalently bound to the binding component (usually the ligand under assay or a binding analog thereof) of the labeled conjugate. The cleaving enzyme employed is a glycosidase which cleaves the bond between the glycone and the dye indicator moiety, releasing a detectable, usually fluorescent, fragment comprising the dye indicator moiety coupled to said binding component (e.g., the ligand). Most preferably, the glycone is a .beta.-galactosyl group and the dye indicator moiety is umbelliferone.